Generation of bioartificial heart tissue by combining a three-dimensional gel-based cardiac construct with decellularized small intestinal submucosa.

The in vitro generation of a bioartificial cardiac construct (CC) represents a promising tool for the repair of ischemic heart tissue. Several approaches to engineer cardiac tissue in vitro have been conducted. The main drawback of these studies is the insufficient size of the resulting construct for clinical applications. The focus of this study was the generation of an artificial three-dimensional (3D), contractile, and suturable myocardial patch by combining a gel-based CC with decellularized porcine small intestinal submucosa (SIS), thereby engineering an artificial tissue of 11 cm² in size. The alignment and morphology of rat neonatal cardiomyocytes (rCMs) in SIS-CC complexes were investigated as well as the re-organization of primary endothelial cells which were co-isolated in the rCM preparation. The ability of a rat heart endothelial cell line (RHE-A) to re-cellularize pre-existing vessel structures within the SIS or a biological vascularized matrix (BioVaM) was determined. SIS-CC contracted spontaneously, uniformly, and rhythmically with an average rate of 200 beats/min in contrast to undirected contractions observed in CC without SIS support. rCM exhibited an elongated morphology with well-defined sarcomeric structures oriented along the longitudinal axis in the SIS-CC, whereas round-shaped and random-arranged rCM were observed in CC. Electric coupling of rCM was demonstrated by microelectrode array measurements. A dense network of CD31⁺/eNOS⁺ cells was detected as permeating the whole construct. Superficial supplementation of RHE-A cells to SIS-CC led to the migration of these cells through the CC, resulting in the re-population of pre-existing vessel structures within the decelluarized SIS. By infusion of RHE-A cells into the BioVaM venous and arterial pedicles, a re-population of the BioVaM vessel bed as well as distribution of RHE-A cells throughout the CC was achieved. Rat endothelial cells within the CC were in contact with RHE-A cells. Ingrowth and formation of a network by endothelial cells infused through the BioVaM represent a promising step toward engineering a functional perfusion system, enabling the engineering of vascularized and well-nourished 3D CC of dimensions relevant for therapeutic heart repair.

Small intestinal submucosa segments as matrix for tissue engineering: review.

Tissue engineering (TE) is an emerging interdisciplinary field aiming at the restoration or improvement of impaired tissue function. A combination of cells, scaffold materials, engineering methods, and biochemical and physiological factors is employed to generate the desired tissue substitute. Scaffolds often play a pivotal role in the engineering process supporting a three-dimensional tissue formation. The ideal scaffold should mimic the native extracellular environment providing mechanical and biological properties to allow cell attachment, migration, and differentiation, as well as remodeling by the host organism. The scaffold should be nonimmunogenic and should ideally be resorbed by the host over time, leaving behind only the regenerated tissue. More than 40 years ago, a preparation of the small intestine was introduced for the replacement of vascular structures. Since then the small intestinal submucosa (SIS) has gained a lot of interest in TE and subsequent clinical applications, as this material exhibits key features of a highly supportive scaffold. This review will focus on the general properties of the SIS and its applications in therapeutical approaches as well as in generating tissue substitutes in vitro. Furthermore, the main problem of TE, which is the insufficient nourishment of cells within three-dimensional, artificial tissues exceeding certain dimensions is addressed. To solve this issue the implementation of another small intestine-derived preparation, the biological vascularized matrix (BioVaM), could be a feasible option. The BioVaM comprises in addition to SIS the arterial and venous mesenteric pedicles and exhibits thereby a perfusable vessel bed that is preserved after decellularization.

A novel miniaturized multimodal bioreactor for continuous in situ assessment of bioartificial cardiac tissue during stimulation and maturation.

Stem cell-based cardiac tissue engineering is a promising approach for regenerative therapy of the injured heart. At present, the small number of stem cell-derived cardiomyocytes that can be obtained using current culture and enrichment techniques represents one of the key limitations for the development of functional bioartificial cardiac tissue (BCT). We have addressed this problem by construction of a novel bioreactor with functional features of larger systems that enables the generation and in situ monitoring of miniaturized BCTs. BCTs were generated from rat cardiomyocytes to demonstrate advantages and usefulness of the bioreactor. Tissues showed spontaneous, synchronized contractions with cell orientation along the axis of strain. Cyclic stretch induced cardiomyocyte hypertrophy, demonstrated by a shift of myosin heavy chain expression from the alpha to beta isoform, together with elevated levels of atrial natriuretic factor. Stretch led to a moderate increase in systolic force (1.42 ± 0.09 mN vs. 0.96 ± 0.09 mN in controls), with significantly higher forces observed after ß-adrenergic stimulation with noradrenalin (2.54 ± 0.11 mN). Combined mechanical and ß-adrenergic stimulation had no synergistic effect. This study demonstrates for the first time that mechanical stimulation and direct real-time contraction force measurement can be combined into a single multimodal bioreactor system, including electrical stimulation of excitable tissue, perfusion of the culture chamber, and the possibility of (fluorescence) microscopic assessment during continuous cultivation. Thus, this bioreactor represents a valuable tool for monitoring tissue development and, ultimately, the optimization of stem cell-based tissue replacement strategies in regenerative medicine.

Engineering a novel three-dimensional contractile myocardial patch with cell sheets and decellularised matrix.

OBJECTIVES: A persistent problem in generating a functional myocardial patch is maintaining contractions in a thicker construct. Thus far, we have successfully created contracting constructs with a defined directionality by seeding neonatal rat cardiomyocytes (CMs) on decellularised porcine small-intestinal submucosa (SIS). Here, we report our efforts in generating a thicker contracting construct by combining CM cell sheets with CM-seeded SIS. METHODS: Porcine SIS was decellularised, opened along the longitudinal axis, fixed in a metal frame (45 mm × 25 mm) and seeded onto the submucosal side with neonatal rat CMs at a density of 1.8 × 10(5) cells cm(-2). CM sheets were prepared using temperature-responsive dishes by seeding CMs at a density of 4.0 × 10(5)cells cm(-2). Three days after CM seeding, one- or three-layered CMs sheet(s) were stacked onto seeded SIS. Construct contraction was observed for an additional 10 days followed by histological analysis. RESULTS: Stacked CM sheets contracted spontaneously and synchronously with seeded SIS after adherence. A large portion of analysed constructs showed a defined contraction direction, parallel to the longitudinal axis (seeded SIS: 83%, seeded SIS+1 sheet: 70%, seeded SIS+3 layered sheets: 71%). This finding was in agreement to the histological finding of aligned CMs parallel to the longitudinal axis. The thickness of seeded SIS with and without three-layered sheets was approximately 800 µm and 500 µm, respectively. CONCLUSIONS: By combining layered CM sheets with CM-seeded SIS, a three-dimensional myocardial patch with contraction in a defined direction was successfully generated. This may represent an intermediate step to a multiple layered, vascularised contractile myocardial graft.

The pro-angiogenic factor CCN1 enhances the re-endothelialization of biological vascularized matrices in vitro.

AIMS: A problem in generating artificial tissues is supplying nutrients to cells within 3D constructs. The use of a decellularized biological vascularized matrix with preserved pedicles (BioVaM), as a scaffold, appears to aid the generation of perfusable tissue constructs in vitro. To prevent vessel occlusion upon implantation, a functional endothelium must line the graft vessel bed. Here we tested whether the pro-angiogenic factor CCN1 could improve the re-endothelialization of BioVaM in vitro. METHODS AND RESULTS: BioVaM vessel beds were incubated with 100 ng/mL recombinant human CCN1. Human cord blood endothelial cells (hCBEC) were analysed with respect to adhesion behaviour upon CCN1 exposure and seeded onto vessel structures of CCN1 exposed BioVaM (cBioVaM). BioVaMs were fixed in a bioreactor and perfusion cultured for 4 and 14 days (d). BioVaM without CCN1 treatment served as controls. Initial seeding success and endothelialization progression were monitored by fluorescence-labelled hCBEC. During construct cultivation, pH and lactate production were measured. Degree of endothelialization and characterization of seeded cells, with respect to endothelial markers, were investigated histologically. BioVaM vessel structures showed a 78 +/- 17% increase of attached cells when pre-treated with CCN1. Evaluation of re-endothelialization (arbitrary units) was 4.0 +/- 0.8 and 2.6 +/- 0.8 after 4 d, and 5.0 +/- 0.0 and 3.0 +/- 0.5 after 14 d in cBioVaM vs. BioVaM, respectively. On day 14, lactate concentration, an indicator of metabolic activity, was increased 12-fold in cBioVaM relative to BioVaM. A preserved endothelial phenotype of seeded cells was verified in all cultures by acetylated low density lipoprotein uptake and positive immunohistochemistry against von Willebrand factor, endothelial nitric oxide synthase, and CD31. CONCLUSION: Coating of decellularized vessel structures with CCN1 supports adhesion of hCBEC and enhances re-endothelialization of BioVaM. Perfusable, endothelialized constructs may aid in solving the problem of nourishing cells inside 3D tissue-engineered constructs.